Project Summary The skeleton relies on a variety of mechanical, biochemical, and hormonal cues to regulate bone strength, structure, and mass. Osteocytes are both the most abundant and most mechanosensitive cells within bone. Located deep in the bone matrix, these cells are optimally positioned to sense and respond to force, directing the activity of other skeletal cells, including osteoblasts and osteoclasts. A variety of molecules influence osteocyte mechanosensation; however, how force is directly transmitted from the mineralized matrix to the cell membrane to induce a biological response remains unknown. We have identified the presence of a complex within osteocytes composed of extracellular perlecan/HSPG2 and a cell surface subunit (a2d1) of voltage sensitive calcium channels (VSCCs). As the perlecan-containing tethers bind the a2d1 subunit of VSCCs, this Matrix-Channel Tethering Complex (M-CTC) enables direct connection between the mineralized matrix and the cell membrane. Thus, the proposed studies will dissect how this mechanosensory complex mechanistically regulates osteocyte behavior, under both basal and mechanical loading conditions. Additionally, the a2d1 subunit of the complex is the receptor for the commonly used antiepileptic and neuropathic pain drug, gabapentin, which can have severe adverse skeletal side effects. Thus, this work will not only investigate the foundational mechanisms through which osteocytes sense force, but will also inform the design of novel strategies to offset the negative effects of gabapentin on bone that reduce bone mass. Our overarching hypothesis is that perlecan-containing tethers, within the M-CTC, transmit force to the a2d1 subunit of VSCCs, enabling mechanical signals to be transduced into anabolic biochemical responses in osteocytes. Aim 1: Determine if osteocyte-specific disruption of the M-CTC membrane receptor a2d1 impairs basal or load-induced bone formation with resultant loss in bone quality. Aim 2: Determine if tissue-specific genetic ablation of the PLN matrix tethers within the M-CTC of osteocytes impairs basal or load-induced bone formation with resultant loss in bone quality. Aim 3: Determine how gabapentin interferes with the M-CTC to impair basal and load-induced bone formation. The function of the M-CTC has not been explored in vivo. As such, these integrated studies, designed to genetically and pharmacologically disrupt the M-CTC and assess the functional consequences, will provide basic insights into the relationship between extracellular tethers and calcium channels in bone. In the long term, this understanding may reduce bone loss in patients treated with gabapentin for neuropathic pain.